Plasmid Purpose:

This plasmid is designed to express tagged proteins in yeast cells (Saccharomyces cerevisiae). The plasmid contains an auxotrophic Uracil selection expression cassette (URA3) that allows for the positive selection of yeast that are deficient in the URA3 gene (YEL021W). This is typically achieved by growing the yeast in minimal media that is reconstituted with the essential amino acids and nucleotides but excluding Uracil.

About the Cleavage Tag:

This plasmid also encodes a protease cleavage site that is designed to be positioned between your gene of interest and the tag to allow the removal of the tag following protein purification or isolation. This plasmid contains a Thrombin cleavage tag. The protein sequence of the cleavage tag is: LVPR?GS. It cleaves preferentially between the Arg and Gly residues. Off target cleavage can often occur at non-specific sites normally from other contaminating proteases. To ensure maximal protein integrity the enzyme reagent must be highly pure.

This plasmid contains the Yeast Elongation Factor Alpha promoter (TEF1). This is the strongest of the yeast promoters that we sell. It is a constitutive promoter and requires no induction. If you are interested in weaker promoters levels than we also stock plasmids that contain the following promoters in order of decreasing strength TPI (strong) ADHI (medium) STE5 (weak). We also stock Galactose inducible promoter plasmids if inducible expression is required. Please contact us for further information.

FLAG is a registered trademark of the Sigma Aldrich Corporation.

About the Peptide Tag:

This plasmid contains a c-terminal Flag epitope tag that can be fused to a gene of interest to allow protein detection and/or purification. The sequence of the tag is: DYKDDDDK

For more information on the methods that can be used to purify proteins please see our protein tag guide.

This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Making Protein Fusions:

This plasmid has been designed to allow three types of cloning into the main MCS to join a coding sequence with the tag.

SnapFusion Cloning:

If you would like to fuse your coding sequence to the tag with minimal additional bases you can use our SnapFusion technology. This process involves amplifying your gene by PCR to add specific restriction sites onto the ends. When these sites are cut they produce an overhang that is compatible with this plasmid cut with BseRI or BsgI.

To insert your gene:

1: Amplify your gene with primers designed using this spreadsheet 2: Cut the plasmid with either BseRI or BsgI.*3: Cut your gene with the enzyme you added using the spreadsheet (any of AcuI BpmI BpuEI BseRI BsgI EciI).4: Clone the gene into the plasmid using DNA ligase.

Using this method with an N-terminal tag plasmid will result in the tag coding sequence immediately followed by your genes ATG start codon at the join. This results in a seamless fusion of the two sequences with no extra bases being added. Using this method on C-terminal tag plasmids will convert your genes stop codon into a TAC (Tyr Y) codon followed by the plasmid tag coding sequence. This results in no extra bases between your gene and the tag. See the diagram below for more information.

*Please note that insect expression plasmids cannot be cut with BsgI only BseRI because of unavoidable conflicting sites in the backbone. Also Yeast plasmids can only be cut with BsgI not BseRI because of conflicting sites in the backbone.

Using this technique will create a gene fragment that can be ligated into any or our >1500 peptide and reporter tag plasmids. If you use one of the other techniques below (Gibson InFusion Seamless or LIC) you will need new primers for every vector you clone into because the arms of homology will change according to the tag plasmid you are cloning into.

If you find that your gene sequence has sites in it that make using this cloning strategy difficult you can still use one of the alternative methods below (e.g. standard cloning or Gibson cloning).

If you are not concerned about leaving a few extra bases between the tag coding sequence and your gene you can clone your gene into the vector using standard cloning restriction enzymes. This strategy will require you to choose which enzymes you want to use to clone your gene.

Open the Primer Design Tool which provides primers with different enzyme choices positioning your gene as close to the tag as possible in each case. Please note that standard enzymes will always leave additional nucleotides between your gene and the tag but using the spreadsheet will ensure the tag and gene are in frame.

These cloning techniques use reagents sold by other companies and allow you to fuse sequences together using enzymes that chew back the DNA to leave overlapping ends/overhangs. The subsequent method of joining the DNA depends on the kit used. To use one of these techniques you can either design your own primers or you can use the spreadsheet below to help with the design.